The ability to genetically transform a plant is useful for studying gene function, producing heterologous proteins, or conferring new properties to the plant such as increased yield or disease resistance. A number of different methods have been developed for introducing transgenes into plants (Veluthambi et al., Current Science 84:368-380, 2003). Generally, each method has three common elements: i) a DNA delivery system, ii) a selection system to differentiate transformed cells or plants from untransformed ones, and iii) a procedure to regenerate the transformed cells or plants. These methods can include in vitro Agrobacterium-mediated gene transfer (tissue culture), in planta Agrobacterium-mediated gene transfer, and physical methods such as microinjection, polyethylene glycol (PEG)-mediated transfer into protoplasts, electroporation of protoplasts, and microprojectile bombardment (biolistics) (Klein et al., Nature 327:70-73, 1987).
Physical methods can be used for certain transformations; however, these methods are limited. For example, in microprojectile, or particle, bombardment, DNA-coated gold particles are introduced into target cells via electric discharge particle acceleration or helium gas. Disadvantages, though, are high copy number and rearrangement of the transgene. Also, with particle bombardment, a tissue culture stage is still necessary, bringing with it the inevitable risk of having somaclonal variation.
Alternatively, in vitro Agrobacterium-mediated gene transfer involves the introduction of a transgene into appropriate plant tissue and regeneration of the tissue into a whole plant. This method has been widely and successfully used with many dicot and monocot crops. However, transformation by tissue culture can be time-consuming and generally very particular to the skills of the researcher performing the transformation. Furthermore, there are several variables that must be considered with this method, such as explant availability, identification of a large population of regenerable cells, accessibility of regenerable cells to Agrobacterium inoculation, and appropriate media and hormones that induce shoot and root regeneration. Since the regeneration of a plant from tissue culture relies upon a few transformed cells, the resulting plants will likely have somaclonal variation, the sum of genetic and epigenetic changes in the transgenic plant that was inherited from the parental cells (Karp, Euphytica 85:295-302, 1995; Larkin and Scowcroft, Theor. Appl. Genet. 60:197-214, 1981).
In contrast, in planta Agrobacterium-mediated gene transfer has advantages over tissue culture or particle bombardment. For example, in planta methods do not require performance by a specialist, and less equipment, labor and reagents are needed to obtain transformed plants. Also, in a given T1 hemizygous transformant, all cells are transgenic. Thus, there is minimal somaclonal variation as compared to that typically encountered with tissue culture (Labra et al., Theor. Appl. Genet. 109:1512-1518, 2004). In planta transformation was first shown with Arabidopsis by imbibing seeds with Agrobacterium (Feldmann and Marks, Mol. Gen. Genet. 208:1-9, 1987). Later, Bechtold et al. (Bechtold et al., C. R. Acad. Sci. (Paris) Life Sci. 316:1194-1199, 1993) demonstrated in planta Agrobacterium-mediated transformation of Arabidopsis using whole plants and vacuum infiltration as a means to increase the likelihood of getting Agrobacterium penetration into the plant (see also, Chang at al., Plant J. 5:551-558, 1994; Mollier et al., C.R. Acad. Sci. (Paris) Life Sci. 318:465-474, 1995; Bechtold and Pelletier, Meth. Mol. Biol. 82:259-266, 1998; Ye et al., Plant J. 19:249-257, 1999; Bechtold et al., Genetics 155:1875-1887, 2000). Vacuum infiltration methods have been used successfully in transforming, for example, pakchoi (Brassica rapa L. ssp. chinensis) (Liu at al., Acta Hortic. 467:187-193, 1998; Qing et al., Mol. Breed. 6:67-72, 2000), alfalfa (Medicago truncatula) (Trieu et al., Plant J. 22:531-541, 2000), Camelina sativa (Lu and Kang, Plant Cell Rep. 27:273-278, 2008, e-pub. September 2007) and Brassica napus (Wang et al., Plant Cell Rep. 22: 274-281, 2003). While transformation has been shown with these particular plant varieties, transformation efficiencies have varied widely. Moreover, the method has not worked for some of the varieties without taking certain, specific steps. For example, vacuum infiltration does not transform Medicago truncatula unless a vernalization treatment is included (Trieu et al., Plant J. 22:531-541, 2000).
More recently, a floral dip method has been developed as an improvement upon in planta Agrobacterium-mediated transformation of Arabidopsis (Clough and Bent, Plant J. 16:735-743, 1998; Clough, Meth. Mol. Biol. 286:91-101, 2005). In the typical floral dip method, a vacuum is no longer required for efficient infiltration of Agrobacterium into the plant. However, frequent multiple applications of dipping solution comprising Agrobacterium to Arabidopsis has been shown to be detrimental to plant health, particularly if the dip intervals are less than every fourth day. Only Arabidopsis and radish (Raphanus sativus L. longipinnatus Bailey) have been successfully transformed by use of a floral dip method (Clough and Bent, Plant J. 16:735-743, 1998; Curtis and Nam, Trans. Res. 10:363-371, 2001; Curtis et al., Trans. Res. 11:249-256, 2002; Curtis, Meth. Mol. Biol. 286:103-110, 2005). A floral dip method has not been found that worked successfully with B. napus (Wang et al., Plant Cell Rep. 22: 274-281, 2003), a crop whose flowers more closely resemble those of Arabidopsis. As such, floral dip methods have only been successful with Arabidopsis and radish, and the transformation technique has been unsuccessful where it has been tried with other plant varieties (Curtis and Nam, Trans. Res. 10:363-371, 2001). These results indicate that floral dip methods are unpredictable as not all plant varieties are transformed with known floral dip techniques. In addition, some plant varieties may be successfully transformed with one technique but not with another.
In light of the current state of plant transformation methods, there remains a need to develop methods that can be used successfully with additional plant varieties. For example, Camelina sativa is an alternative oilseed crop whose oil holds promise for use in industrial applications, nutrition, and biofuels. Thus, there would be value in developing transformation systems for this crop to enable manipulation of its agronomic qualities. A transformation system for Camelina sativa via tissue regeneration has been described (WO 02/38779 A1). In addition, Lu and Kang (Plant Cell Rep. 27:273-278, 2008, e-pub. September 2007) recently reported in planta Agrobacterium-mediated transformation of Camelina using a vacuum infiltration method. However, they were not able to obtain transformants by floral dip without vacuum infiltration. Therefore, despite these recently developed transformation systems for Camelina sativa, better and less complex techniques need to be explored.
Accordingly, the present invention provides methods of transforming Camelina sativa plants that offer unique advantages over currently existing techniques.